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Athletes usually arrange their training plans and determine their training intensity according to the coach’s experience and simple physical indicators such as heart rate during exercise. However, the accuracy of this method is poor, and the training plan and exercise intensity arranged according to this method can easily cause physical damage, or the training cannot meet the actual needs. Therefore, in order to realize the reasonable arrangement and monitoring of athletes’ training, a method of human exercise intensity recognition based on ECG (electrocardiogram) and PCG (Phonocardiogram) is proposed. First, the ECG and PCG signals are fused into a two-dimensional image, and the dataset is marked and divided according to the different motion intensities. Then, the training set is trained with a CNN (convolutional neural network) to obtain the prediction model of the neural network. Finally, the neural network model is used to identify the ECG and PCG signals to judge the exercise intensity of the athlete, so as to adjust the training plan according to the exercise intensity. The recognition accuracy of the model on the dataset can reach 95.68%. Compared with the use of heart rate to detect the physical state during exercise, ECG records the total potential changes in the process of depolarization and repolarization of the heart, and PCG records the waveform of the beating sound of the heart, which contains richer feature information. Combined with the CNN method, the athlete’s exercise intensity prediction model constructed by extracting the features of the athlete’s ECG and PCG signals realizes the real-time monitoring of the athlete’s exercise intensity and has high accuracy and generalization ability.

Reasonable exercise is beneficial to human health. However, it is difficult for ordinary athletes to judge whether they are already in a state of fatigue that is not suitable for exercise. In this case, it is easy to cause physical damage or even life-threatening. Therefore, to health sports, protecting the human body in sports not be injured by unreasonable sports, this study proposes an exercise fatigue diagnosis method based on short-time Fourier transform (STFT) and convolutional neural network (CNN). The method analyzes and diagnoses the real-time electrocardiogram, and obtains whether the current exerciser has exercise fatigue according to the electrocardiogram. The algorithm first performs short-time Fourier transform on the electrocardiogram (ECG) signal to obtain the time spectrum of the signal, which is divided into training set and validation set. The training set is then fed into the convolutional neural network for learning, and the network parameters are adjusted. Finally, the trained convolutional neural network model is applied to the test set, and the recognition result of fatigue level is output. The validity and feasibility of the method are verified by the ECG experiment of exercise fatigue degree. The experimental recognition accuracy rate can reach 97.70%, which proves that the constructed sports fatigue diagnosis model has high diagnostic accuracy and is feasible for practical application.

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

Depression is a mental illness and can even lead to suicide if not be diagnosed and treated. Electroencephalograph (EEG) is used to diagnose depression and it is more complexity to extract the features from all the multimodal channel information . In order to simplify the diagnose process and detect clinical depression, the EEG channels with strong depression information should be identified firstly. Therefore, a depression signal correlation identification method based on convolutional neural network (CNN) is proposed. In the method, the labeled multi-channel EEG is used as data. The EEG signals of each channel are divided into neural network training data set and these data is trained by AlexNet network. Then the correlation classification of each channel for depression is identified based on the trained sample. Accuracy and loss functions are used to evaluate classification index.Conversely, the correlation is lower. An experiments is conducted and the results show that the correlation is not consistent. A few of channels are strongly correlated with depression, such as 13, 17, 28, 40, 46, 66 and 69. These EEG channels are selected to diagnose depression.

The gearbox is an important component in the mechanical transmission system and plays a key role in aerospace, wind power and other fields. Gear failure is one of the main causes of gearbox failure, and therefore it is very important to accurately diagnose the type of gear failure under different operating conditions. Aiming at the problem that it is difficult to effectively identify the fault types of gears using traditional methods under complex and changeable working conditions, a fault diagnosis method based on multi-sensor information fusion and Visual Geometry Group (VGG) is proposed. First, the power spectral density is calculated with the raw frequency domain signal collected by multiple sensors before being transformed into a power spectral density energy map after information fusion. Second, the obtained energy map is combined with VGG to obtain the fault diagnosis model of the gear. Finally, two datasets are used to verify the effectiveness and generalization ability of the method. The experimental results show that the accuracy of the method can reach 100% at most on both datasets.

Generative adversarial nets (GANs) have been successfully applied to the artificial generation of image data. In terms of text data, much has been done on the artificial generation of natural language from a single corpus. We consider multiple text corpora as the input data, for which there can be two applications of GANs: (1) the creation of consistent cross-corpus word embeddings given different word embeddings per corpus; (2) the generation of robust bag-of-words document embeddings for each corpora. We demonstrate our GAN models on real-world text data sets from different corpora, and show that embeddings from both models (weGAN and deGAN) lead to improvements in supervised learning problems.

Skin disease is a common diseases. Although it is very common, the manifestations of skin diseases are various, and the diagnosis process is relatively difficult, and the requirements for dermatologists are also very high. And the recognition efficiency of detection also needs to be improved. To this end, this paper mainly investigates the potential of classifying skin diseases in deep learning convolutional neural networks to enhance the recognition accuracy. The VGG16 neural network was trained using the training data of the ISIC 2019 challenge, and the training data was augmented to increase the accuracy of the model, and accurately classify 3 skin diseases with 97% accuracy. This method can help doctors to make auxiliary diagnosis and provide technical support for skin disease diagnosis.

Unreasonable exercise will cause damage to the body. In physical education, coaches only use physiological indicators such as heart rate and breathing to judge the physiological state of athletes, which is highly subjective and is not conducive to accurately judging the physiological state of athletes. In order to effectively monitor athletes in exercises, a method for identifying athletes' exercise intensity based on ECG and convolutional neural network was proposed. In this method, the more informative ECG signal is used as the physiological indicator of the athlete's exercise intensity, combined with the convolutional neural network for feature extraction, and finally the training model is used to monitor and evaluate the athlete's exercise intensity. The method implements automatic feature extraction and recognition of athletes' ECG signals. The simulation results of the dataset show that the method can effectively judge the exercise intensity, and the accuracy can reach 98.6%. At the same time, the algorithm has a small amount of calculation and a fast convergence speed, in the daily training of athletes has a good auxiliary role.

In order to solve the problem of AGV fault detection system’s complexion and low accuracy, a convolutional neural network (CNN) based on the status monitoring and fault diagnosis method for automatic guided vehicle (AGV) is proposed. Firstly, the vibration signals of the core components of AGV are converted into two-dimensional (2D) images. Secondly, 2D images are input into convolution neural network for training. Finally, the trained model is used to monitor the running status of AGV and identify faults. The results show that the proposed method can effectively monitor the status of AGV in operation.

Epilepsy, as a common nervous system disease in the world, urgently needs effective methods for diagnosis and treatment. Scalp electroencephalogram (EEG) contains a lot of physiological and pathological information, which plays a very important role in the diagnosis of brain diseases such as epilepsy. At present, the clinical analysis of EEG signal is mainly based on the clinician’s visual analysis, which makes the clinician’s task heavy, and there is no quantitative standard for the analysis results. Therefore, the automatic classification of epileptic EEG signals has great potential in current clinical application. Based on the deep learning VGG16 convolutional neural network model, a method for automatic detection of seizure signals is proposed in this paper. Firstly, the samples are preprocessed, then enter the VGG16 convolutional neural network model for training, and finally input the validation set for validation. The experimental results indicate that this model can distinguish the pre-ictal EEG, interictal EEG and ictal EEG.

Identifying stroke during a 9-1-1 call is critical to timely prehospital care. However, emergency medical dispatchers (EMDs) recognize stroke in less than half of 9-1-1 calls, potentially due to the words used by callers to communicate stroke signs and symptoms. We hypothesized that callers do not typically use words and phrases considered to be classical descriptors of stroke, such as focal neurologic deficits, but that a mixed-methods approach can identify words and phrases commonly used by 9-1-1 callers to describe acute stroke victims.We performed a mixed-method, retrospective study of 9-1-1 call audio recordings for adult patients with confirmed stroke who were transported by ambulance in a large urban city. Content analysis, a qualitative methodology, and computational linguistics, a quantitative methodology, were used to identify key words and phrases used by 9-1-1 callers to describe acute stroke victims. Because a caller's level of emotional distress contributes to the communication during a 9-1-1 call, the Emotional Content and Cooperation Score was scored by a multidisciplinary team.A total of 110 9-1-1 calls, received between June and September 2013, were analyzed. EMDs recognized stroke in 48% of calls, and the emotional state of most callers (95%) was calm. In 77% of calls in which EMDs recognized stroke, callers specifically used the word "stroke"; however, the word "stroke" was used in only 38% of calls. Vague, non-specific words and phrases were used to describe stroke victims' symptoms in 55% of calls, and 45% of callers used distractor words and phrases suggestive of non-stroke emergencies. Focal neurologic symptoms were described in 39% of calls. Computational linguistics identified 9 key words that were more commonly used in calls where the EMD identified stroke. These words were concordant with terms identified through qualitative content analysis.Most 9-1-1 callers used vague, non-specific, or distractor words and phrases and infrequently provide classic stroke descriptions during 9-1-1 calls for stroke. Both qualitative and quantitative methodologies identified similar key words and phrases associated with accurate EMD stroke recognition. This study suggests that tools incorporating commonly used words and phrases could potentially improve EMD stroke recognition.

Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR–Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction–modification (R–M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R–M system. This study might shed light on exploiting and improving CRISPR–Cas systems in other microorganisms for genome editing and metabolic engineering practices.

Background/purpose It is important to confirm product use effects on skin health for products intended for prolonged skin contact. This study compared experimental and marketed reference adult incontinence protective underwear. Methods Randomized, single-blind (examiner), parallel study evaluating skin health effects in predominantly obese incontinent women normally using protective underwear (approximately 20% Type II Diabetes). Subjects wore experimental or marketed reference protective underwear daily, 14 consecutive days. Visual skin grading, transepidermal water loss (TEWL) assessed before, after 1 and 2 weeks of product wear. Overall assessment of comfort assessed. Results Of the 122 subjects (60 experimental and 62 marketed reference), 22 were diabetic and 88 were postmenopausal. Under the conditions of this study, there were no statistically significant differences in overall change from baseline for visual grading and TEWL between the experimental product and the marketed reference product for all subjects. Changes from baseline for skin erythema and skin marking were generally small for both products for all subjects as well as for both diabetics and non-diabetics. There were no serious adverse events (AEs), and no withdrawals due to AEs. Overall comfort assessments of size and fit were 'just right,' and skin comfort in the leg, waist and crotch areas were 'comfortable' or 'very comfortable' for both products. Conclusions In-use 14-day testing demonstrated few statistical differences between experimental product with unique odor neutralizing technology and currently marketed product for skin assessments and comfort. Both products were comfortable and well-tolerated.

The chemical kinetics on gelatin–CdS direct conjugates has been systematically investigated as a function of different temperature and reactant concentration (i.e. Cd2+, S2− and gelatin) by UV–visible spectroscopy, for the first time. The nonlinear fitting and the differential method were used to calculate the initial rate based on the absorbance–time data. A double logarithmic linear equation for calculating the rate constant (k) and the reaction order (n) was introduced. The reaction kinetic parameters (n, k, Ea, and Z) and activation thermodynamic parameters (ΔG≠, ΔH≠, and ΔS≠) were obtained from variable temperature kinetic studies. The overall rate equation allowing evaluation of conditions that provide required reaction rate could be expressed as: r = 1.11 × 10 8 exp ( - 4971 / T ) [ Cd 2 + ] [ gelatin ] 0.6 [ S 2 - ] 0.6 ( M / s ) The calculated values of the reaction rate are well coincide with the experimental results. A suitable kinetic model is also proposed. This work will provide guidance for the rational design of gelatin-directed syntheses of metal sulfide materials, and help to understand the biological effects of nanoparticles at the molecular level.

Water-soluble gelatin-PbS bionanocomposites (BNCs) were synthesized via a facile one-pot chemical reaction method at pH 7.40. The samples were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), UV-vis absorption spectra (UV-vis), Fourier transform infrared spectra (FT-IR) and circular dichroism (CD). FT-IR data were used to envisage the binding of PbS particles with oxygen atoms of carbonyl groups of gelatin molecule. The possible integration mechanism between gelatin and PbS was discussed in detail. The effect of Pb2+ and PbS on the conformations of gelatin has also been analyzed by means of UV-vis, CD and FT-IR spectra, resulting in less α-helix content and more open structures (β-sheet, β-turn, or expanded). A new formula to calculate the association constant was proposed according to the relationship between the absorbance of gelatin-PbS BNCs and the free concentration of PbS, and apparent association constants K (298/303/308 K: 3.11/2.00/1.60 × 106 mol/L) at three different temperatures were calculated based on this formula. Thermodynamic parameters such as ΔGθ, ΔHθ and ΔSθ were also determined. The results of the thermodynamic investigations indicated that the reaction was spontaneous (ΔGθ < 0), and enthalpy-driven (ΔHθ < 0).

Manufacturers of consumer products consistently seek to improve marketed products in terms of both safety and efficacy. The desire for continued improvement is seen even in well-established products such as catamenial products which have existed in some form for thousands of years. A recent innovation in the design of menstrual pads is the addition of a surface finish of emollient for the purpose of increasing comfort during wear. The present paper presents different evaluations of such an emollient-treated menstrual pad with a novel absorbent core. These investigations demonstrated product tolerability, defined the optimal formulation and concentration of the emollient-containing finish, and demonstrated successful transfer of the emollient to the relevant skin surface. In addition, enhancement of skin moisturization, associated with exposure to the emollient-treated pad, was demonstrated by several technologies: assessment of skin moisturization by Corneometer®, skin friction testing, and skin capacitance.

Apertured polyethylene films (AF) have been used as a surface for sanitary pads for decades because they are compatible with the skin and keep the pad surface drier. A modified film (AF-plus) with improved fluid handling and a smoother, suppler texture has been developed. We hypothesized that these changes would improve both performance and skin compatibility. However, distinguishing the skin effects of materials that are inherently mild is a challenge.(i) To compare the skin irritation potential of pads with AF-plus modified film relative to the standard AF film and (ii) to assess the potential for the AF-plus film to induce delayed contact hypersensitivity.Pads bearing the AF-plus film were compared to pads with the standard AF film in a behind-the-knee (BTK) test, which assesses the combination of chemical irritation and frictional effects of materials applied to the popliteal fossa under a semi-occlusive bandage. Erythema on the skin surface was scored with the naked eye and subsurface tissue erythema was visualized and scored using cross-polarized illumination. Skin dryness was scored with the naked eye only. One-sided statistical evaluations were performed to test the hypothesis of AF-plus film superiority. The potential of the AF-plus film to induce delayed contact hypersensitivity was assessed by a human repeat insult patch test (HRIPT).Pads with the AF-plus surface were significantly milder to skin in the BTK test, producing lower levels of both surface and subsurface tissue erythema. Moreover, subjects with preexisting erythema on the skin surface at study start developed comparatively less erythema over the course of the study overall with the AF-plus pad compared to the AF pad. No significant difference in skin dryness was observed between product groups. The AF-plus pad showed no evidence of inducing delayed contact hypersensitivity.The AF-plus pad was superior to the AF pad in terms of skin mildness as discerned by objectively scored surface and subsurface tissue erythema. In subjects with preexisting erythema, the AF-plus pad appeared to contribute less to the further development of inflammation under the test conditions. Given the compositional similarities in the two films, the results could point to more limited contribution of the AF-plus film to skin friction, one of the factors simulated by the BTK test protocol.

We have developed a line of products designed to better meet the overall needs of women suffering from urinary incontinence. The products are more discrete and contain a unique odor neutralizing technology (ONT).This paper describes the overall skin compatibility program for this product line in which the new products were compared to negative controls and/or commercially marketed reference products with an established history of safe use.Test products consisted of several product forms (light pads/pantiliners, moderate pads, briefs and taped diapers) with ONT and having various degrees of protection. Studies were conducted using standard protocols for 4-day and 21-day cumulative irritation, the Human Repeat Insult Patch Test (HRIPT), and the Behind-the-Knee (BTK) test for mechanical and chemical irritation. In one 4-day irritation study and one HRIPT, test subjects consisted of individuals with self-assessed sensitive skin. In addition, one 4-day study was conducted using normal skin sites, and sites compromised by tape stripping. Nonirritant controls were physiologic saline and/or current, commercially marketed incontinence products. All responses were evaluated by visual scoring of erythema. In addition, in the BTK, transepidermal water loss (TEWL) and adverse sensory effects collected from panelists' daily diaries were also evaluated.Two 4-day cumulative irritation studies and one 21-day study demonstrated that a wide range of product forms (liners, light and moderate pads, briefs and adult diapers) produced skin reactions similar to the nonirritant controls. The 4-day study conducted using sensitive skin subjects showed good skin compatibility, and the test products were comparable to the nonirritant controls. In the 4-day study with both normal and compromised skin sites, test products produced mean erythema scores similar to the nonirritant controls. Three HRIPT separate studies confirm that the products do not induce contact sensitization, including one study conducted on individuals with self-assessed sensitive skin. In the BTK, test and control products produced similar irritation, as assessed by erythema, TEWL and sensory effects.The results from the patch tests and mechanical irritation test demonstrate good skin compatibility of the new line of products with the unique ONT. In addition, the forms of the product (i.e. liner, pad or brief), were equally compatible with skin.

Fibroblast growth factor receptor 4 (FGFR4) is a member of the FGFR family of receptor tyrosine kinases, and plays important roles in a variety of biological functions such as cell proliferation, differentiation, migration, angiogenesis, tissue repair, and tumorigenesis. The human FGFRs share a high degree of sequence homology between themselves, as well as with their murine homologs. Consequently, it has been suggested that it may be difficult to prepare monoclonal antibodies (MAbs) that are specific for the individual receptor types. In this communication, we report on the development and characterization of a panel of anti-human FGFR4 MAbs that were generated in mice using a rapid immunization protocol. Using a modified rapid immunization at multiple sites (RIMMS) protocol with the soluble extracellular domain of human FGFR4 (FGFR4-ECD), the immunized mice developed high levels of polyclonal IgG to the immunogen within 13 days of the first immunization. The lymph node cells isolated from the immunized animals were then fused with mouse myeloma cells for hybridoma generation. Use of an efficient hybridoma cloning protocol in combination with an ELISA screening procedure allowed for early identification of stable hybridomas secreting antihuman FGFR4 IgG. Several identified MAbs specifically reacted with the FGFR4 protein without binding to the other human isoforms (FGFR1, FGFR2, and FGFR3). As evaluated by BIAcore analysis, most anti-FGFR4 MAbs displayed high affinities (8.6 x 10(8) approximately 3.9 x 10(10) M) to FGFR4. Furthermore, these MAbs were able to bind to FGFR4 expressed on human breast tumor cell lines MDA-MB-361 and MDA-MB-453. Taken together, the results demonstrate that the RIMMS strategy is an effective approach for generating class-switched, high-affinity MAbs in mice to evolutionarily conserved proteins such as human FGFR4. These MAbs may be useful tools for further investigation of the biological functions and pathological roles of human FGFR4.

Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data. We systematically investigated which vector design most effectively displays and expresses Fab molecules in E. coli using, as a model system, a human Fab against tetanus toxoid (tt). Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively. The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E. coli were compared for the aforementioned vectors. Our results showed that the pFab3 vector was most effective in Fab display. A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2. Further experiments were conducted using pFab3 to optimize expression levels using different strains of E. coli and various culture conditions. The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.

The effect of pressure on excited-state intramolecular proton transfer (ESIPT) is studied in three polyhydroxyflavones: 3,7-dihydroxyflavone, 3,5,7-trihydroxyflavone (galangin), and 3,3',4',7-tetrahydroxy-flavone (fisetin), dissolved in poly(methyl methacrylate) (PMMA), poly(vinyl acetate) (PVAc), poly(acrylic acid) (PAA), and cellulose acetate (CAC). The unusual photophysical properties of polyhydroxyflavone in PAA are interpreted in terms of protonation of the carbonyl oxygen. We calculate the ratio of the equilibrium constant at elevated pressure to that at 1 atm, K (p)/K (0), and the difference of partial molar volume between the normal state and the phototautomer, ΔV. The results indicate that pressure effectively suppresses intramolecular proton transfer in polyhydroxyflavone. K (p)/K (0) and ΔV show a strong pressure dependence in the range of 10-30 kbar.

Infection of a human melanoma cell line by a retroviral vector resulted in transmission of a mouse VL30 (mVL30-1) retroelement RNA to some of the cells infected by the retrovirus, followed by synthesis, integration, and expression of the mVL30-1 cDNA. One vector carried a tissue factor (TF) transgene that generated high TF melanoma clones, and another vector was a control without the TF transgene that generated low TF clones. Some high TF melanoma clones contained the mVL30-1 retroelement and others did not, and some low TF melanoma clones contained the mVL30-1 retroelement and others did not. Each type of melanoma clone was tested for its metastatic potential in severe combined immunodeficient (SCID) mice, by i.v. injection of the cells to generate lung tumors. None of the low TF clones that either contained or lacked the mVL30-1 retroelement generated lung tumors, consistent with earlier results showing that high TF expression promoted metastasis. The high TF clones containing the mVL30-1 retroelement were strongly metastatic, in contrast to the high TF clones lacking the mVL30-1 retroelement, which were weakly metastatic. Southern hybridization analyses showed that the mVL30-1 cDNA integrated into different genomic sites in different melanoma clones, suggesting that the effect of the mVL30-1 retroelement on metastasis depends not on integration per se but instead on expression of the mVL30-1 RNA. A role for the mVL30-1 RNA in metastasis and possibly other cell functions is an unexpected finding, because the RNA appears to lack significant coding potential for a functional protein. The metastatic effect might be mediated directly by a noncoding mVL30-1 RNA or by a peptide or small protein encoded by one of the short ORFs in the mVL30-1 RNA.

Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary andDrosophilaS2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. Anin vitrocytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. Thein vitroresults suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumorsin vivo.

To understand the molecular mechanism of gibberellin-dependent gene regulation, the effect of three phosphatase inhibitors on the germination of rice seeds and the expression of a target gene, the α-amylase gene, Osamy-c, were measured. We found that okadaic acid, microcystin-LR, and calyculin A, which are known to specifically inhibit Ser/Thr phosphatases 1 and 2A, strongly inhibit the expression of the Osamy-c and may be involved in the germination of rice seeds.

Advances in measuring devices have made it feasible to conveniently measure localized changes in skin temperature. A series of experiments were conducted to determine if the addition of surface skin temperature measurements into studies commonly used to screen for skin responses to consumer products would increase the ability to discriminate between similar, mild products. Three testing protocols were evaluated: the modified arm patch with 24-h occlusive exposure for 4 consecutive applications, the behind the knee (BTK) with 6-h occlusive exposures with pressure for 5 consecutive days, and the bikini area shaving study with assessments after manual shaving by women with self-reported irritation after using a manual razor. In the modified arm patch studies, the positive irritant control (0.1 % sodium lauryl sulfate) produced mean erythematous scores that were higher (p < 0.05) than reactions produced by the negative irritant control (0.9 % saline), or the mild products. The skin surface temperatures were not significantly different in any arm patch study. In the BTK the two mild products produced significantly different levels of erythema at both the afternoon scoring (30–60 min after sample removal) and the morning scoring (after approximately 18 h of recovery). Skin surface temperatures differed only at the morning scoring. In the bikini area shaving study, mean erythema after 48 h was lower (p < 0.05) than after 5 min or 24 h on the left side only, indicating some degree of handedness. Skin surface temperature was significantly higher prior to the shaving exercise than at any of the subsequent scoring time points. Results indicate that the addition of measurements of skin surface temperature using DermaTemp® to standard exposure protocols used to evaluate potential skin responses to consumer products would not increase the ability to discriminate between products.

The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I beta-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the beta-sheet of the N-terminal subdomain. The type I beta-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this beta-turn has little effect on EGF-receptor interactions.

In this report we describe the production of a [Lys3,Tyr22] murine epidermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N-succinimidyl)-[15N,2H16]doxyl-2-spiro-4'-pimelate ([15N,2H16]BSSDP) in order to study the rotational dynamics of the EGF/EGF receptor complex by saturation-transfer electron paramagnetic resonance (ST-EPR). Previous results [Faulkner-O'Brien et al. (1991) Biochemistry 30,8976-8985] indicated that the reaction of [15N,2H16]BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of the EGF/EGF receptor complex because the major product, in which [15N,2H16]BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the more tightly coupled bidentate product was unstable. Using oligonucleotide-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for [15N,2H16]BSSDP labeling. The [Lys3,Tyr22] mEGF was expressed in Escherichia coli HB101 transformed with a pIN-III-ompA3-[Lys3,Tyr22] mEGF plasmid and was purified from the bacterial periplasm using a simple two step purification method. The [15N,2H16]BSSDP reacted with [Lys3,Tyr22]mEGF in high yield, and EPR analysis of the major product revealed tight motional coupling between the spin probe and the protein. Biological activity, as assessed by stimulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label. The [15N,2H16]BSSDP-modified [Lys3,Tyr22] mEGF was shown to be equipotent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF binding to the EGF receptor in A431 membrane vesicles. Using the [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR.

Background: Type 2 diabetes (T2), previously a disease of adult onset, is increasing in prevalence amongst children and young adults. Yet the presence of cardiovascular risks, such as left ventricular hypertrophy (LVH) are largely unknown in this population. Purpose: To determine the frequencies of abnormal left ventricular (LV) mass and geometry in young people with T2. Methods: LV mass (LVM) was assessed by echo, indexed to height in meters 2.7, and compared to healthy Obese (O) and Lean (L) controls of similar age, gender matched, non-diabetics. Statistical analysis was performed with non-parametric tests. Data are presented as mean and standard deviations. Results: A total of 98 T2 (ages 11–23 yrs, 19 ± 4 yrs; 64% female, 36% male; BMI 35 ± 8) were evaluated and compared to O & L controls (BMI 36 ± 7 and 21 ± 3, respectively). Although indexed LVM was higher for T2 compared to L (36 ± 11 vs. 27 ± 6 gm/m2.7, p = ). Systolic (SBP) and diastolic (DBP) blood pressure were higher in the T2 vs. O & L (p Conclusions: Young people with T2 have higher indexed LVM and abnormal LV geometry compared to lean controls. Obesity, not fasting plasma glucose seems to be a primary factor associated with LVM. These data suggest that obesity is a primary factor in the early cardiac changes in youth with T2. Early weight loss may have a greater beneficial effect on cardiac geometry than glycemic control. Table I: Prevalence of Left Ventricular Geometries

ObjectivesThe purpose of this study was to evaluate changes in cardiac geometry, systolic and diastolic function before and after weight loss in morbidly obese adolescents.BackgroundCardiac abnormalities are present in morbidly obese adolescents; however, it is unclear if they are reversible with weight loss.MethodsData from 38 adolescents (13 to 19 years; 29 females, 9 males, 33 Caucasians, 5 African Americans) were evaluated before and after bariatric surgery. Left ventricular mass (LVM), left ventricular (LV) geometry, systolic and diastolic function were assessed by echocardiography. Mean follow up was 10 ± 3 months.ResultsWeight and body mass index decreased post-operatively (mean weight loss 59 ± 15 kg, pre-operative body mass index 60 ± 9 kg/m2 vs. follow-up 40 ± 8 kg/m2, p < 0.0001). Change in LVM index (54 ± 13 g/m2.7 to 42 ± 10 g/m2.7, p < 0.0001) correlated with weight loss (r = 0.41, p = 0.01). Prevalence of concentric left ventricular hypertrophy (LVH) improved from 28% at pre-operative to only 3% at follow up (p = 0.007), and normal LV geometry improved from 36% to 79% at follow up (p = 0.009). Diastolic function also improved (mitral E/Ea lateral 7.7 ± 2.3 at pre-operative vs. 6.3 ± 1.6 at post-operative, p = 0.003). In addition, rate-pressure product improved suggesting decreased cardiac workload (p < 0.001).ConclusionsElevated LVM index, concentric LVH, altered diastolic function, and cardiac workload significantly improve following surgically induced weight loss in morbidly obese adolescents. Large weight loss due to bariatric surgery improves predictors of future cardiovascular morbidity in these young people.

Tissue factor (TF) is a type I transmembrane protein and the initiator of the extrinsic blood coagulation pathway. TF plays a critical role in tumor development and its overexpression is observed in many tumors. To understand the prevalence and relative level of TF expression in non-small-cell lung cancer (NSCLC), we analyzed 50 NSCLC tumors by immunohistochemical staining and found that 88% of human NSCLC tumors overexpressed TF. We then generated a high affinity anti-TF antibody, TF278, which specifically binds TF on the surface of cells and is internalized upon binding. An 111In-labeled TF278 demonstrated favorable tumor accumulation in an SW-900 xenograft tumor model with a maximum mean percent of injected dose per gram of tissue (%ID/g) of 73.1% at 96 hours postinjection. In addition, we labeled the antibody with 90Y and tested its ability to inhibit the growth of tumors in an SW-900 xenograft tumor model in immunocompromised mice. The 90Y-TF278 slowed the growth of SW-900 tumors at a 50 microCi dose and completely regressed SW-900 tumors at a 150 microCi dose with little toxicity.

Tissue factor (TF) plays an important, physiological role in hemostasis. Recent studies have demonstrated the over-expression of TF in a number of solid tumor types and its pathological roles in angiogenesis and tumor metastasis. In this study, we report the development and characterization of a panel of murine MAbs that are specific for human TF, but do not inhibit TF-mediated blood coagulation. By using a modified repetitive immunizations at multiple sites (RIMMS) protocol in conjunction with an efficient hybridoma cloning procedure, anti-TF MAbs were generated within a relatively short time frame of 5-6 weeks. Following primary screening by ELISA, the binding of the MAbs to the native form of human TF was demonstrated in flow cytometry using a stable cell line expressing human TF. Several of these TF-specific MAbs did not inhibit blood coagulation in a blood coagulation assay and bound with high affinity (0.5-2 nM) to human TF in BIAcore analyses. Importantly, this study represents an independent evaluation of the RIMMS strategy for MAb generation and demonstrates that class-switched, high-affinity MAbs can be generated rapidly and reliably using RIMMS.

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The β-glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.

A ubiquitin-conjugating enzyme (UBC) gene, induced by gibberellin (GA) within an hour, was identified in rice (Oryza sativa) seeds by the mRNA differential display technique. GA inducibility was confirmed by RNA hybridization. A full-length UBC cDNA clone and a genomic clone have been isolated and sequenced. The deduced amino acid sequence shares a significant identity with several known UBC sequences, which are probably involved in the pathway responsible for degrading short-lived regulatory proteins. In vivo transient assays using the UBC gene promoter, joined to the luciferase cDNA as the reporter gene, showed that the sequence located between positions 231 and 159 upstream of the transcription start site of this promoter is crucial for GA-dependent activation of the luciferase cDNA. Finally, trans-activation experiments indicated that this UBC gene is involved in gibberellin-stimulated alpha-amylase gene expression.

The ultimate goal of this investigation was to identify intermediary steps in the gibberellin (GA)-dependent signaling pathway in rice aleurone cells. By using a differential display approach, a number of putative GA-responsive genes were isolated. One of them, a GA-responsive Ca(2+)-ATPase gene, was identified and partially characterized. A genomic clone and a cDNA clone were isolated and sequenced. The deduced amino acid sequence showed that this protein resembles an endoplasmic reticulum membrane Ca(2+)-ATPase. In a transient assay in rice aleurone cells, expression of the introduced Ca(2+)-ATPase cDNA bypassed the GA requirement for stimulating the expression of a major target gene, the alpha-amylase c gene (Osamy-c). This result suggests that GA-dependent expression of this Ca(2+)-ATPase gene (OsCa-atpase) plays an important role in the GA-dependent signal-transduction pathway. To investigate the possible involvement of other proteins and genes that may affect the intracellular Ca2+ level, compounds which can block different putative steps in the signal-transduction pathway were introduced into rice aleurone cells, and then the level of the OsCa-atpase transcript or the Osamy-c transcript was monitored. In the presence of GA, the rice Ca(2+)-ATPase and the Ca2+ channels appeared to co-regulate the local concentration of cytosolic Ca2+. The release of Ca2+ from the internal stores to the cytoplasm was presumably initiated by inositol-1,4,5-triphosphate which reached a peak level within 25 min after GA induction. As a second messenger, Ca2+ binds to calmodulin (CaM), and the Ca2+/CaM complex regulates the cytosolic Ca2+ by affecting expression of the OsCa-atpase. Finally, a working model is proposed for the GA-dependent signaling pathway in aleurone cells.